The transition from preosteoblasts to osteoblasts involves the secretion of a complex extracellular matrix containing bone morphogenetic proteins, growth factors and structural components that interact with cells to induce osteoblast-specific gene expression. MARK signaling is known to be critical for the responsiveness of osteoblasts extracellular matrix, hormonal, growth factor and mechanical stimuli. As shown by this laboratory, differentiation is controlled in part by binding of type I collagen to a2p1 integrins that signal to the nucleus via the ERK/MAPK pathway to phosphorylate and activate the osteoblast-related transcription factor, RUNX2. Other factors such as FGF2, IGF-1 and mechanical stimulation, known to signal through MARK, also stimulate RUNX2 phosphorylation. Consensus MARK phosphorylation sites were identified in RUNX2, two of which were shown to be critical for MARK responsiveness. The significance of MARK signaling to osteoblast differentiation was established by showing that perturbation of this pathway through the use of specific inhibitors or expression of constitutively active or dominant negative MARK intermediates, respectively, increased or decreased osteoblast differentiation in cell culture and in transgenic mice. In this renewal, we will pursue these important findings by addressing the following aims: Aim 1. Establish the importance of MARK phosphorylation to the biological activity of RUNX2 in cell culture and in vivo. Hypothesis: Consensus MARK phosphorylation sites at amino acids 301 and 319 of RUNX2 are critical for responsiveness to MARK signals. Studies will: a) evaluate the role of MARK phosphorylation sites in ECM/BMP-induced osteoblast differentiation and response of osteoblasts to FGF2, IGF-1 and mechanical stimulation in cell culture, b) examine the role of phosphorylation in the regulation of RUNX2 transcriptional activity, c) evaluate the importance of RUNX2 phosphorylation in vivo using a knock- in approach, d) initiate studies to identify additional MARK and other phosphorylation sites in RUNX2 and new nuclear factors interacting with RUNX2 in a phosphorylation-dependent manner. Aim 2. Evaluate the role of the MARK activity in osteoblast/osteocyte function in adult and ageing mice. Hypothesis: MARK activity is necessary for normal osteoblast function during adult life and will mediate the response of bone to various challenges associated with ageing, remodeling after ovarectomv and mechanical loading. Proposed studies will use recently developed transgenic mice expressing cpnstitutively-active and dominant-negative MARK intermediates in osteoblasts to evaluate the role of MARK signaling in adult mice as follows: a) An ageing study will compare the bone properties in transgenic and wild type mice through adult life and senescence, b) An ovarectomy model will be used to evaluate the role of MARK signaling in the remodeling skeleton, c) Mechanoresponsiyeness: wild type, TgMek-sp and TgMek-dn mice will be subjected to mechanical loading and the bone formation response determined. [unreadable] These studies will define the role of RUNX2 phosphorylation and the ERK/MAPK pathway in osteoblast differentiation, bone formation and remodeling. Mechanisms defined in these studies have the potential to explain how bone responds to a number of extracellular stimuli including ECM and mechanical signals and may serve as the basis for new therapeutic approaches targeting MARK signaling in bone. [unreadable] [unreadable] [unreadable]